1. Field of the Invention
The present invention relates to the field of isolation and purification of biological molecules. More specifically, the present invention pertains to methods, compositions, and kits for separating and purifying nucleic acids.
2. Description of Related Art
Sulfolane is a clear, colorless liquid commonly used in the chemical industry as an extractive distillation solvent or reaction solvent (see FIG. 1). It was originally developed as a solvent to purify butadiene (see, for example, U.S. Pat. No. 4,090,923). Sulfolane has several applications in molecular biology. It has been used along with other liquids to form an upper and lower phase in a method to extract nucleic acids (see, for example, U.S. Pat. No. 6,815,541). It has also been employed to improve cell lysis and solubilization in a process for rapid isolation of high molecular weight DNA (see, for example, U.S. Pat. No. 4,900,677). Furthermore, it can be used as an aprotic solvent to deactivate ribonucleases and denature proteins in general (see, for example, U.S. Pat. No. 5,637,687). In addition, it has been reported that it can also be used to enhance PCR (see, for example, U.S. Pat. No. 6,949,368).
Isolation of biological molecules, such as DNA and RNA, and their subsequent analysis is a fundamental part of molecular biology. Analysis of nucleic acids is crucial to gene expression studies, not just in basic research, but also in the medical field of diagnostic use. For example, diagnostic tools include those for detecting nucleic acid sequences from minute amounts of cells, tissues, and/or biopsy materials, and for detecting viral nucleic acids in blood or plasma. The yield and quality of the nucleic acids isolated and purified from a sample has a critical effect on the success of any subsequent analyses.
Isolation of nucleic acids from a biological sample usually involves lysing the biological sample by, for example, mechanical action and/or chemical action followed by purification of the nucleic acids. Previously, purification of nucleic acids was performed using methods such as cesium chloride density gradient centrifugation (which is time-consuming and expensive) or extraction with phenol (which is considered unhealthy for the user). In a typical final step, ethanol precipitation was used to concentrate the nucleic acids, which resulted in lower yields of the isolated nucleic acids.
Many of the methods currently used to isolate nucleic acids are based on the adsorption of the nucleic acid on glass or silica particles in the presence of a chaotropic salt (see, for example, Vogelstein, B. and Gillespie, D., “Preparative and analytical purification of DNA from agarose”, Proc. Natl. Acad. Sci. USA 76:615-619, 1979; U.S. Pat. Nos. 5,234,809; and 6,180,778).
However, methods that allow easy isolation of nucleic acids, proteins, and other molecules from various cells and tissues, that have improved yields, that provide better quality of isolated product, and that do not require organic solvent extraction or ethanol precipitation are still needed in the art.